1. Fitness Trade-Offs Associated with the Evolution of Resistance to Antifungal Drug Combinations 
Jessica A. Hill, Teresa R. O’Meara, Leah E. Cowen 
Cell Reports 
Volume 10, Issue 5, Pages (February 2015)
DOI: /j.celrep
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2. Cell Reports 2015 10, 809-819DOI: (10.1016/j.celrep.2015.01.009)
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3. Figure 1
Lab-Passaged Strains and Late-Stage Clinical Isolates of C. Albicans Evolved Resistance to Drug Combinations
(A) Of the seven experimentally evolved strains, six are resistant to FL and FK506, and one is resistant to FL and GdA.
(B) A series of clinical isolates recovered over 2 years from a single patient have a two-step increase in resistance to drug combinations. Subsequent analyses focus on key isolates reflecting the two-step increase in resistance to these drug combinations (CaCi-2, CaCi-12, CaCi-13, CaCi-15, CaCi-16, and CaCi-17), indicated by the pink triangles.
(C and D) Fitness of in vitro (C) and in vivo (D) evolved strains was assessed relative to their respective ancestors in FL and GdA (top) and FL and FK506 (bottom). Generally, in vitro evolved strains passaged in FL and FK506 (Chr4(2n+1), Chr4,R(2n+1), Chr4,7(2n+1), Chr4,5R,6,7(2n+1), LCB1L390F, CNA1S401Stop) are more fit in that environment but not in FL and GdA and vice versa (HSP90D91Y was passaged in FL and GdA). In vivo-evolved strains increase in fitness in both FL and GdA and FL and FK506 over time, despite not having experienced these drug combinations in the host. Susceptibility assays were performed in duplicate at 30°C for 48 hr, where optical densities were standardized to no drug control wells.
Data in (C) and (D) are represented as mean ± SEM. ∗p < 0.05, ∗∗p < See also Table S1.
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4. Figure 2
FL Resistance of Clinical Isolates Is Largely Hsp90 and Calcineurin Independent, in Contrast to In Vitro-Evolved Strains
(A) Transcriptional repression of HSP90 abrogates FL resistance of the earliest clinical isolate, CaCi-2, while only minimally decreasing FL resistance in the last isolate, CaCi-17.
(B) Rendering calcineurin nonfunctional by deletion of its catalytic subunit, CNA1, abrogates FL resistance in CaCi-2 but has only a minor effect on FL resistance in CaCi-17.
(C) Deleting CNA1 in the background of four in vitro-evolved strains abrogates FL resistance, in contrast to the clinical isolates. Deleting CNA1 in the background of LCB1L390F does not, however, indicating that FL resistance in this strain is calcineurin independent. Susceptibility assays performed in duplicate in YPD at 37°C for 48 hr (A and B) or at 30°C for 24 hr (C). Optical densities were standardized to no drug control wells.
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5. Figure 3
Together, TAC1A736V, UPC2A643V, and ERG11R467K Confer Resistance to FL with GdA or FK506
(A–C) TAC1A736V, UPC2A643V, and ERG11R467K were placed in the background of CaCi-2 individually or in combinations that reflect genotypes found in later clinical isolates concurrent with increases in resistance to drug combinations (UPC2A643V/UPC2 ERG11R467K/ERG11R467K in CaCi-13; UPC2A643V/UPC2 ERG11R467K/ERG11R467K TAC1A736V/TAC1 in CaCi-17). Resistance to FL was assessed as a single agent (A) or in combination with GdA (B) or FK506 (C). Individual mutations confer a modest increase in resistance to FL and FK506, although no effect on resistance to FL and GdA. Recapitulating the genotype of the last clinical isolate, CaCi-17, confers resistance to FL and GdA and FL and FK506 to nearly the same FL concentration as found in CaCi-17. Susceptibility assays performed in duplicate in YPD at 37°C for 48 hr. Optical densities were standardized to no drug control wells. TAC1∗ = TAC1A736V; UPC2∗ = UPC2A643V; ERG11∗ = ERG11R467K.
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6. Figure 4
Resistance to Drug Combinations Is Costly in the Absence of Drug and in Different Stressful Environments
(A–G) Fitness relative to the ancestor was measured for in vitro and in vivo evolved lineages by competitive fitness assays in (A) the absence of drug, (B) FL alone, (C) the cell wall stress CFW, (D) the cell membrane stress SDS, (E) salt stress, (F) the oxidative stress H2O2, and (G) heat stress. Each circle represents the mean of three replicate competition experiments, with standard error bars. Differences in fitness between strains were determined by ANOVAs with Bonferroni corrections for multiple comparisons and subsequent Tukey post hoc tests. Data represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.
(H and I) Clustering strains evolved in vitro (H) or in vivo (I) by fitness, standardized to the ancestor.
See also Figure S1.
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7. Figure 5
Evolved Strains Are More Readily Killed by Macrophages Than the Ancestor
Strains were incubated with J774A.1 macrophages for 1 hr before macrophages were lysed and viable C. albicans cells quantified by plating for CFUs. Differences between strains were determined by ANOVAs with Bonferroni corrections for multiple comparisons and subsequent Tukey post hoc tests. Data represented as mean ± SD. ∗p < 0.05; ∗∗p < 0.01.
See also Figure S2.
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8. Figure 6
Late-Stage Clinical Isolates Do Not Filament in Response to GdA
(A) In vitro evolved strains grow as yeast in 30°C, but filament in response to 39°C and 10% serum at 37°C in YPD after 6 hr.
(B) Clinical isolates grow as yeast in 30°C, but filament in response to 39°C and 10% serum at 37°C. Late-stage isolates are unable to filament in the response to 10 μM GdA at 30°C, unlike early-stage isolates. Strains were grown in YPD for 6 hr. Scale bar represents 10 μm.
See also Figure S3.
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9. Figure 7
TAC1A736V Abolishes Filamentation in Late-Stage Clinical Isolates
(A) Transcriptional repression of HSP90 in both the first and last clinical isolate induces filamentation. All strains were grown in YPD with a low concentration of doxycycline (  μg/ml) for 18 hr at 30°C, for transcriptional repression of HSP90 in the tetO- HSP90 strains.
(B) TAC1A736V confers resistance to GdA. After 6 hr in YPD with 10 μM GdA at 30°C, CaCi-2 grows filamentously, while CaCi-17 grows as yeast. The ability to undergo morphogenesis is abolished in CaCi-2 when a WT copy of TAC1 is replaced by TAC1A736V in that background. Scale bar represents 20 μm.
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